FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023952935> ?p ?o ?g. }

• W2023952935 endingPage "15166" @default.
• W2023952935 startingPage "15161" @default.
• W2023952935 abstract "The CTP:glycerol-3-phosphate cytidylyltransferase (GCT) of Bacillus subtilis has been shown to be similar in primary structure to the CTP:phosphocholine cytidylyltransferases of several organisms. To identify the residues of this cytidylyltransferase family that function in catalysis, the conserved hydrophilic amino acid residues plus a conserved tryptophan of the GCT were mutated to alanine. The most dramatic losses in activity occurred with H14A and H17A; these histidine residues are part of an HXGH sequence similar to that found in class I aminoacyl-tRNA synthetases. The k cat values for H14A and H17A were decreased by factors of 5 × 10−5and 4 × 10−4, respectively, with no significant change in K m values. Asp-11, which is found near the HXGH sequence in the cytidylyltransferases but not aminoacyl-tRNA synthetases, was also important for activity, with the D11A mutation decreasing activity by a factor of 2 × 10−3. Several residues found in the sequence RTEGISTT, a signature sequence for this cytidylyltransferase family, as well as other isolated residues were also shown to be important for activity, with k cat values decreasing by factors of 0.14–4 × 10−4. The K m values of three mutant enzymes, D38A, W74A, and D94A, for both CTP and glycerol-3-phosphate were 6–130-fold higher than that of the wild-type enzyme. Mutant enzymes were analyzed by two-dimensional NMR to determine if the overall structures of the enzymes were intact. One of the mutant enzymes, D66A, was defective in overall structure, but several of the others, including H14A and H17A, were not. These results indicate that His-14 and His-17 play a role in catalysis and suggest that their role is similar to the role of the His residues in the HXGH sequence in class I aminoacyl-tRNA synthetases,i.e. to stabilize a pentacoordinate transition state. The CTP:glycerol-3-phosphate cytidylyltransferase (GCT) of Bacillus subtilis has been shown to be similar in primary structure to the CTP:phosphocholine cytidylyltransferases of several organisms. To identify the residues of this cytidylyltransferase family that function in catalysis, the conserved hydrophilic amino acid residues plus a conserved tryptophan of the GCT were mutated to alanine. The most dramatic losses in activity occurred with H14A and H17A; these histidine residues are part of an HXGH sequence similar to that found in class I aminoacyl-tRNA synthetases. The k cat values for H14A and H17A were decreased by factors of 5 × 10−5and 4 × 10−4, respectively, with no significant change in K m values. Asp-11, which is found near the HXGH sequence in the cytidylyltransferases but not aminoacyl-tRNA synthetases, was also important for activity, with the D11A mutation decreasing activity by a factor of 2 × 10−3. Several residues found in the sequence RTEGISTT, a signature sequence for this cytidylyltransferase family, as well as other isolated residues were also shown to be important for activity, with k cat values decreasing by factors of 0.14–4 × 10−4. The K m values of three mutant enzymes, D38A, W74A, and D94A, for both CTP and glycerol-3-phosphate were 6–130-fold higher than that of the wild-type enzyme. Mutant enzymes were analyzed by two-dimensional NMR to determine if the overall structures of the enzymes were intact. One of the mutant enzymes, D66A, was defective in overall structure, but several of the others, including H14A and H17A, were not. These results indicate that His-14 and His-17 play a role in catalysis and suggest that their role is similar to the role of the His residues in the HXGH sequence in class I aminoacyl-tRNA synthetases,i.e. to stabilize a pentacoordinate transition state. The CTP:glycerol-3-phosphate cytidylyltransferase (GCT) 1The abbreviations used are: GCT, CTP:glycerol-3-phosphate cytidylyltransferase; CCT, CTP:phosphocholine cytidylyltransferase; ECT, CTP:phosphoethanolamine cytidylyltransferase; IPTG, isopropyl-1-thio-β-d-galactopyranoside; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; NTA, nitrilotriacetic acid; HSQC, heteronuclear single quantum correlation.1The abbreviations used are: GCT, CTP:glycerol-3-phosphate cytidylyltransferase; CCT, CTP:phosphocholine cytidylyltransferase; ECT, CTP:phosphoethanolamine cytidylyltransferase; IPTG, isopropyl-1-thio-β-d-galactopyranoside; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; NTA, nitrilotriacetic acid; HSQC, heteronuclear single quantum correlation. from Bacillus subtiliscatalyzes the formation of CDP-glycerol and pyrophosphate from CTP and glycerol-3-phosphate. CDP-glycerol then serves as a principal precursor required for biosynthesis of poly(glycerol phosphate), the major teichoic acid found in the bacterial cell wall (1Mauel C. Young M. Karamata D. J. Gen. Microbiol. 1991; 137: 929-941Crossref PubMed Scopus (73) Google Scholar, 2Pooley H.M. Abellan F.-X. Karamata D. J. Gen. Microbiol. 1991; 137: 921-928Crossref PubMed Scopus (47) Google Scholar). GCT appears to be a member of a cytidylyltransferase family that includes CTP:phosphocholine cytidylyltransferases (CCT), a key regulatory enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis in higher eukaryotes (3Kent C. Prog. Lipid Res. 1990; 29: 87-105Crossref PubMed Scopus (158) Google Scholar, 4Vance D.E. Vance D.E. Phosphatidylcholine Metabolism. CRC Press, Inc., Boca Raton, FL1989: 33-45Google Scholar, 5Bork P. Holm L. Koonin E.V. Sander C. Proteins. 1995; 22: 259-266Crossref PubMed Scopus (88) Google Scholar). Fig. 1 shows a comparison of the deduced amino acid sequences of several cytidylyltransferases: presumed GCTs from Staphylococcus aureus andStreptomyces wedmorensis, GCT from B. subtilis, CCTs from a variety of organisms, and ethanolamine phosphate cytidylyltransferase (ECT) from yeast. This comparison reveals a number of residues that are conserved among these three enzymes (6Park Y.S. Sweitzer T.D. Dixon J.E. Kent C. J. Biol. Chem. 1993; 268: 16648-16654Abstract Full Text PDF PubMed Google Scholar). These conserved amino acids are contained within the catalytic core of the CCTs (7Kalmar G.B. Kay R.J. Lachance A. Aebersold R. Cornell R.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6029-6033Crossref PubMed Scopus (129) Google Scholar). Conservation of these amino acids suggests that they may be required for functions such as catalysis, recognition of substrates, structural integrity, or association of subunits.Considering the high degree of similarity of GCT with CCT and ECT, it would be useful to use the GCT as a model cytidylyltransferase for studying structure-function relationships. We have expressed the GCT in Escherichia coli, purified it in large quantities, and examined its physicochemical characteristics (6Park Y.S. Sweitzer T.D. Dixon J.E. Kent C. J. Biol. Chem. 1993; 268: 16648-16654Abstract Full Text PDF PubMed Google Scholar). The availability of recombinant GCT expressed in E. coli permits the use of site-directed mutagenesis to search for catalytically important amino acids. Site-directed mutagenesis has allowed direct, quantifiable assessment of the contributions of individual amino acids toward enzyme specificity and catalysis.In the present study, conserved hydrophilic residues, a conserved tryptophan, and a non-conserved cysteine residue were chosen for mutagenesis. The mutated GCTs were expressed and purified as histidine-tagged forms and subjected to kinetic and structural analyses to evaluate the contributions of those residues in the GCT to catalytic parameters. The CTP:glycerol-3-phosphate cytidylyltransferase (GCT) 1The abbreviations used are: GCT, CTP:glycerol-3-phosphate cytidylyltransferase; CCT, CTP:phosphocholine cytidylyltransferase; ECT, CTP:phosphoethanolamine cytidylyltransferase; IPTG, isopropyl-1-thio-β-d-galactopyranoside; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; NTA, nitrilotriacetic acid; HSQC, heteronuclear single quantum correlation.1The abbreviations used are: GCT, CTP:glycerol-3-phosphate cytidylyltransferase; CCT, CTP:phosphocholine cytidylyltransferase; ECT, CTP:phosphoethanolamine cytidylyltransferase; IPTG, isopropyl-1-thio-β-d-galactopyranoside; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; NTA, nitrilotriacetic acid; HSQC, heteronuclear single quantum correlation. from Bacillus subtiliscatalyzes the formation of CDP-glycerol and pyrophosphate from CTP and glycerol-3-phosphate. CDP-glycerol then serves as a principal precursor required for biosynthesis of poly(glycerol phosphate), the major teichoic acid found in the bacterial cell wall (1Mauel C. Young M. Karamata D. J. Gen. Microbiol. 1991; 137: 929-941Crossref PubMed Scopus (73) Google Scholar, 2Pooley H.M. Abellan F.-X. Karamata D. J. Gen. Microbiol. 1991; 137: 921-928Crossref PubMed Scopus (47) Google Scholar). GCT appears to be a member of a cytidylyltransferase family that includes CTP:phosphocholine cytidylyltransferases (CCT), a key regulatory enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis in higher eukaryotes (3Kent C. Prog. Lipid Res. 1990; 29: 87-105Crossref PubMed Scopus (158) Google Scholar, 4Vance D.E. Vance D.E. Phosphatidylcholine Metabolism. CRC Press, Inc., Boca Raton, FL1989: 33-45Google Scholar, 5Bork P. Holm L. Koonin E.V. Sander C. Proteins. 1995; 22: 259-266Crossref PubMed Scopus (88) Google Scholar). Fig. 1 shows a comparison of the deduced amino acid sequences of several cytidylyltransferases: presumed GCTs from Staphylococcus aureus andStreptomyces wedmorensis, GCT from B. subtilis, CCTs from a variety of organisms, and ethanolamine phosphate cytidylyltransferase (ECT) from yeast. This comparison reveals a number of residues that are conserved among these three enzymes (6Park Y.S. Sweitzer T.D. Dixon J.E. Kent C. J. Biol. Chem. 1993; 268: 16648-16654Abstract Full Text PDF PubMed Google Scholar). These conserved amino acids are contained within the catalytic core of the CCTs (7Kalmar G.B. Kay R.J. Lachance A. Aebersold R. Cornell R.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6029-6033Crossref PubMed Scopus (129) Google Scholar). Conservation of these amino acids suggests that they may be required for functions such as catalysis, recognition of substrates, structural integrity, or association of subunits. Considering the high degree of similarity of GCT with CCT and ECT, it would be useful to use the GCT as a model cytidylyltransferase for studying structure-function relationships. We have expressed the GCT in Escherichia coli, purified it in large quantities, and examined its physicochemical characteristics (6Park Y.S. Sweitzer T.D. Dixon J.E. Kent C. J. Biol. Chem. 1993; 268: 16648-16654Abstract Full Text PDF PubMed Google Scholar). The availability of recombinant GCT expressed in E. coli permits the use of site-directed mutagenesis to search for catalytically important amino acids. Site-directed mutagenesis has allowed direct, quantifiable assessment of the contributions of individual amino acids toward enzyme specificity and catalysis. In the present study, conserved hydrophilic residues, a conserved tryptophan, and a non-conserved cysteine residue were chosen for mutagenesis. The mutated GCTs were expressed and purified as histidine-tagged forms and subjected to kinetic and structural analyses to evaluate the contributions of those residues in the GCT to catalytic parameters. The authors thank Joel Clement, Jon Friesen, and James Peliska for helpful discussions." @default.
• W2023952935 created "2016-06-24" @default.
• W2023952935 creator A5009411980 @default.
• W2023952935 creator A5013358867 @default.
• W2023952935 creator A5018238937 @default.
• W2023952935 creator A5065487329 @default.
• W2023952935 creator A5084076127 @default.
• W2023952935 creator A5085131424 @default.
• W2023952935 date "1997-06-01" @default.
• W2023952935 modified "2023-10-18" @default.
• W2023952935 title "Identification of Functional Conserved Residues of CTP:glycerol-3-phosphate Cytidylyltransferase" @default.
• W2023952935 cites W1576342917 @default.
• W2023952935 cites W1929550892 @default.
• W2023952935 cites W1949275432 @default.
• W2023952935 cites W1972635679 @default.
• W2023952935 cites W1984547849 @default.
• W2023952935 cites W1985204165 @default.
• W2023952935 cites W1986719399 @default.
• W2023952935 cites W1991923790 @default.
• W2023952935 cites W2007487901 @default.
• W2023952935 cites W2019449559 @default.
• W2023952935 cites W2021320387 @default.
• W2023952935 cites W2025488960 @default.
• W2023952935 cites W2034708139 @default.
• W2023952935 cites W2043988433 @default.
• W2023952935 cites W2047043554 @default.
• W2023952935 cites W2052007204 @default.
• W2023952935 cites W2059157388 @default.
• W2023952935 cites W2069734146 @default.
• W2023952935 cites W2100837269 @default.
• W2023952935 cites W2101439753 @default.
• W2023952935 cites W2104732374 @default.
• W2023952935 cites W2108092566 @default.
• W2023952935 cites W2132417094 @default.
• W2023952935 cites W2138270253 @default.
• W2023952935 cites W2636914 @default.
• W2023952935 cites W4293247451 @default.
• W2023952935 cites W56444061 @default.
• W2023952935 doi "https://doi.org/10.1074/jbc.272.24.15161" @default.
• W2023952935 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9182537" @default.
• W2023952935 hasPublicationYear "1997" @default.
• W2023952935 type Work @default.
• W2023952935 sameAs 2023952935 @default.
• W2023952935 citedByCount "68" @default.
• W2023952935 countsByYear W20239529352012 @default.
• W2023952935 countsByYear W20239529352013 @default.
• W2023952935 countsByYear W20239529352014 @default.
• W2023952935 countsByYear W20239529352015 @default.
• W2023952935 countsByYear W20239529352016 @default.
• W2023952935 countsByYear W20239529352017 @default.
• W2023952935 countsByYear W20239529352018 @default.
• W2023952935 countsByYear W20239529352019 @default.
• W2023952935 countsByYear W20239529352021 @default.
• W2023952935 crossrefType "journal-article" @default.
• W2023952935 hasAuthorship W2023952935A5009411980 @default.
• W2023952935 hasAuthorship W2023952935A5013358867 @default.
• W2023952935 hasAuthorship W2023952935A5018238937 @default.
• W2023952935 hasAuthorship W2023952935A5065487329 @default.
• W2023952935 hasAuthorship W2023952935A5084076127 @default.
• W2023952935 hasAuthorship W2023952935A5085131424 @default.
• W2023952935 hasConcept C116834253 @default.
• W2023952935 hasConcept C185592680 @default.
• W2023952935 hasConcept C18903297 @default.
• W2023952935 hasConcept C2777132085 @default.
• W2023952935 hasConcept C55493867 @default.
• W2023952935 hasConcept C70721500 @default.
• W2023952935 hasConcept C86803240 @default.
• W2023952935 hasConceptScore W2023952935C116834253 @default.
• W2023952935 hasConceptScore W2023952935C185592680 @default.
• W2023952935 hasConceptScore W2023952935C18903297 @default.
• W2023952935 hasConceptScore W2023952935C2777132085 @default.
• W2023952935 hasConceptScore W2023952935C55493867 @default.
• W2023952935 hasConceptScore W2023952935C70721500 @default.
• W2023952935 hasConceptScore W2023952935C86803240 @default.
• W2023952935 hasIssue "24" @default.
• W2023952935 hasLocation W20239529351 @default.
• W2023952935 hasOpenAccess W2023952935 @default.
• W2023952935 hasPrimaryLocation W20239529351 @default.
• W2023952935 hasRelatedWork W2001960069 @default.
• W2023952935 hasRelatedWork W2033505165 @default.
• W2023952935 hasRelatedWork W2057457746 @default.
• W2023952935 hasRelatedWork W2101226880 @default.
• W2023952935 hasRelatedWork W2134702784 @default.
• W2023952935 hasRelatedWork W2616533555 @default.
• W2023952935 hasRelatedWork W2950717927 @default.
• W2023952935 hasRelatedWork W2951805389 @default.
• W2023952935 hasRelatedWork W2952273270 @default.
• W2023952935 hasRelatedWork W1578520212 @default.
• W2023952935 hasVolume "272" @default.
• W2023952935 isParatext "false" @default.
• W2023952935 isRetracted "false" @default.
• W2023952935 magId "2023952935" @default.
• W2023952935 workType "article" @default.